Monoclonal antibody against VP2 protein of mandarin fish disegmental RNA virus: preparation method and application

Abstract

This invention provides a monoclonal antibody against the VP2 protein of a mandarin fish two-segmented RNA virus, its preparation method, and its application. The VP2 protein was obtained by expression and purification using recombinant plasmids. Mice were immunized three times, and mice with the highest serum titer were selected for intraperitoneal booster immunization against VP2 protein. Mouse spleen cells and SP2 / 0 myeloma cells were fused, and hybridoma cell lines secreting VP2 protein antibodies were screened. These cells were then subcloned to obtain four stable hybridoma cell lines secreting anti-VP2 protein antibodies, and their heavy and light chain variable region sequences were determined. Mice were injected intraperitoneally with Freund's incomplete adjuvant, followed by the injection of the hybridoma cell lines. Ascites fluid was collected from the mice, extracted, purified, and used to obtain the monoclonal antibody against the VP2 protein. Using these four monoclonal antibodies, three highly conserved and specific antigenic epitopes of the VP2 protein of the mandarin fish two-segmented RNA virus were screened and identified. This invention provides support for establishing specific serological methods for mandarin fish two-segmented RNA viruses and for further research on the biological function and clinical diagnosis of VP2.

Description

Technical Field

[0001] This invention belongs to the technical field of bioengineering, specifically relating to a monoclonal antibody against the VP2 protein of a two-segmented RNA virus in mandarin fish, its preparation method, and its application. Background Technology

[0002] Siniperca chuatsi Birnavirus (SCBV) is a two-segmented RNA virus first isolated and identified from mandarin fish in 2023. Infected mandarin fish exhibit symptoms such as loss of appetite, lethargy, and unsteady swimming, with a final mortality rate exceeding 90%. Autopsy reveals open mouths, varying degrees of hemorrhage on the surface, and ulcers on some fish. Dissection reveals fluid in the abdominal cavity, yellowish viscous material in the intestines, an enlarged spleen, and a discolored liver with white necrosis.

[0003] Double RNA viruses are a class of viruses with a two-segmented, double-stranded RNA genome. The Bisnaviridae family comprises seven genera, including *Avibirnavirus*, *Aquabirnavirus*, *Entomobirnavirus*, *Blosnavirus*, *Dronavirus*, *Ronavirus*, and *Telnavirus*. Important members of this family include infectious bursal disease virus and infectious pancreatic necrosis virus. The mandarin fish double-segmented RNA virus belongs to the Bisnaviridae family and is classified under the genus *Blosnavirus*. This non-enveloped virus is approximately 60 nm in diameter and has an icosahedral structure. Its genome consists of two segments of double-stranded RNA; segment A encodes three proteins, VP2, VP3, and VP4, while segment B encodes the VP1 protein.

[0004] The genome of the dual RNA virus encodes multiple proteins, including VP2 (38 kDa) and VP3 (33 kDa), which are structural proteins constituting the viral capsid. VP2 and VP3 are the outer and inner capsid proteins, respectively. VP3 contains group-specific antigens, but VP2 is the only structural protein capable of inducing neutralizing antibodies and exhibits extremely high antigen affinity. VP2 contains a major immunodominant antigen domain, carrying the main targets for protective and neutralizing antibodies. The high immunogenicity of the VP2 protein makes it an ideal target for use as a diagnostic antigen in immunoassays. In-depth research on the virus, understanding its characteristics and infection mechanisms, will help develop targeted diagnostic methods and control strategies. Since the mandarin fish dual-segment RNA virus is a newly emerging virus, there is currently no specific treatment for mandarin fish infected with this virus. Farmers urgently need a simple, convenient, and rapid detection method for early diagnosis and control.

[0005] Therefore, the monoclonal antibody against the VP2 protein of mandarin fish two-segment RNA virus prepared in this invention, and the identification of its specific antigenic epitopes and variable region sequences, lay the foundation for in-depth research on mandarin fish two-segment RNA virus and the development of diagnostic kits. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a monoclonal antibody against the VP2 protein of mandarin fish bisegmental RNA virus (SCBV), its preparation method, and its application, thereby resolving the issues in the background art. The method of this invention mainly studies the VP2 protein and prepares a monoclonal antibody against SCBV VP2 protein, which has the advantages of high titer, strong affinity, and good specificity, providing a foundation for the study of the structure and function of SCBV VP2 protein.

[0007] On one hand, this invention provides the following technical solution: a method for preparing a monoclonal antibody against the VP2 protein of a two-segmented RNA virus in mandarin fish, the method comprising: S1. The VP2 gene of SCBV was amplified and cloned into pET-28a(+) to obtain the recombinant plasmid pET28a-VP2. Then, the recombinant plasmid pET28a-VP2 was transformed into competent cells for expression and purified to obtain the VP2 protein. S2. The purified VP2 protein was emulsified with Freund's adjuvant and immunized with 6-week-old female BALB / c mice. After immunization, blood was collected from the tail vein of the mice and serum was separated. The antibody titer of the immunized mice was detected by indirect ELISA. The mice with the highest titer were selected for intraperitoneal injection of VP2 protein for booster immunization. S3. Spleen cells from mice after booster immunization were collected and fused with SP2 / 0 myeloma cells. When the confluence of fused cells reached more than 50%, positive hybridoma cells secreting VP2 antibody were screened by indirect ELISA. The positive hybridoma cells were subcloned to obtain a hybridoma cell line that stably secretes monoclonal antibody against mandarin fish two-segment RNA virus VP2 protein. S4. Eight-week-old female BALB / c mice were selected and injected intraperitoneally with Freund's incomplete adjuvant. Seven days later, the hybridoma cell line described above was injected, at a dose of 1×10⁻⁶. 6 Each mouse was tested until its abdomen swelled, then ascites was extracted, centrifuged, and purified to obtain a monoclonal antibody against the VP2 protein of the mandarin fish two-segment RNA virus.

[0008] The beneficial effects of this invention are as follows: This invention prepared purified VP2 protein with high protein content and good purity, and a protein concentration of 1.2 mg / mL; This invention prepared four monoclonal antibodies, namely 2-1, 13-1, 20-1, and 22-1. The heavy chain of antibody 2-1 is IgG2b, and the heavy chains of antibodies 13-1, 20-1, and 22-1 are IgG1. The light chains of all antibodies are Kappa. The antibodies prepared by this invention have the advantages of high titer, strong affinity, and specificity. This invention utilizes a monoclonal antibody against the VP2 protein of a two-segmented RNA virus in mandarin fish to identify three antigenic epitopes of the VP2 protein. These epitopes exhibit high conservation and specificity, laying a solid foundation for conducting functional studies of the VP2 protein and establishing a highly sensitive detection method.

[0009] In addition, the method for preparing monoclonal antibodies against the VP2 protein of mandarin fish bisegmental RNA virus provided by the present invention may also have the following additional technical features: Preferably, in step S1, the step of transforming the recombinant plasmid pET28a-VP2 into competent cells for expression specifically includes: The recombinant plasmid pET28a-VP2 was transformed into BL21(DE3) competent cells, and single colonies were picked and cultured. The bacterial culture was then inoculated into LB medium containing kanamycin resistance and cultured with shaking until OD. 600 When the concentration was 0.6, IPTG solution was added to induce protein expression; The conditions for culturing single colonies were as follows: overnight culture at 37℃ and 220 rpm; the conditions for inoculating the bacterial culture into LB medium containing kanamycin resistance and shaking culture were as follows: the bacterial culture was inoculated into LB medium containing kanamycin resistance at a ratio of 1:100 and cultured at 37℃ and 220 rpm for 4 hours with shaking; the conditions for inducing protein expression by adding IPTG solution were as follows: protein expression was induced by adding IPTG solution to a final concentration of 1 mM and cultured at 37℃ and 220 rpm for 6 hours.

[0010] Preferably, in step S1, the purification step specifically includes: Collect the bacterial cells after expression culture, resuspend the cells in PBS, sonicate and centrifuge to collect the precipitate, lyse with lysis buffer and collect the supernatant solution for column chromatography purification.

[0011] Preferably, in step S2, the immunization of mice specifically involves: The purified VP2 protein was emulsified with Freund's complete adjuvant at a 1:1 ratio at 100 μg / mouse and injected subcutaneously at multiple sites on the back of mice for immunization. Two weeks later, VP2 protein was emulsified with Freund's incomplete adjuvant at a 1:1 ratio for a second immunization. Two weeks later, a third immunization was performed. One week later, blood was collected from the tail vein of the immunized mice.

[0012] Preferably, in step S2, the step of detecting the antibody titer in the serum of immunized mice using an indirect ELISA method specifically includes: 100 ng / well of purified VP2 protein was coated onto an ELISA plate overnight at 4°C. The plate was then blocked with 5% skim milk powder at 37°C for 2 h. The plate was washed with PBST buffer. Serum from immunized mice was diluted proportionally and used as the primary antibody. The plate was incubated at 37°C for 1 h. The plate was washed with PBST buffer. HRP-labeled goat anti-mouse antibody diluted 1:5000 was used as the secondary antibody and incubated at 37°C for 30 min. The plate was washed with PBST buffer. 100 μL / well of chromogenic solution was added and the plate was incubated at 37°C for 10 min. Finally, 50 μL / well of stop solution was added, and the data for each well were read using a microplate reader.

[0013] Preferably, in step S3, the specific steps for fusing spleen cells from mice after booster immunization with SP2 / 0 myeloma cells are as follows: After booster immunization, spleen cells from mice were fused with SP2 / 0 cells at a ratio of 5:1. 50% PEG1450 was added after 1 min, and the mixture was stirred for 2 min. 10 mL of preheated culture medium was added within 10 min. The mixture was incubated at 37°C for 5 min, centrifuged at 200 x g for 7 min, and then mixed into HAT medium containing 20% ​​FBS. 100 μL / well was added to 96-well dishes pre-coated with feeder cells until the confluence of cells to be successfully fused reached more than 50%.

[0014] Preferably, in step S3, the subcloning of positive hybridoma cells specifically involves performing four subclonings using a limiting dilution method to obtain four hybridoma cell lines with antibodies against the VP2 protein of the mandarin fish two-segment RNA virus.

[0015] On the other hand, the invention provides the following technical solution: a monoclonal antibody against the VP2 protein of a mandarin fish bisegmental RNA virus, wherein the monoclonal antibody against the VP2 protein of a mandarin fish bisegmental RNA virus is prepared by the preparation method of the monoclonal antibody against the VP2 protein of a mandarin fish bisegmental RNA virus as described above.

[0016] On the other hand, the invention provides the following technical solutions, such as the application of the above-mentioned monoclonal antibody against the bisegmental RNA virus VP2 protein of mandarin fish in identifying the antigenic epitopes of VP2 protein and preparing SCBV detection and diagnostic products.

[0017] Preferably, when the monoclonal antibody against the bisegmental RNA virus VP2 protein of mandarin fish is monoclonal antibody 2-1, the antigenic epitope region of the VP2 protein it identifies is 1-25 aa; when the monoclonal antibody against the bisegmental RNA virus VP2 protein of mandarin fish is monoclonal antibody 13-1, the antigenic epitope region of the VP2 protein it identifies is 280-300 aa; when the monoclonal antibody against the bisegmental RNA virus VP2 protein of mandarin fish is monoclonal antibody 20-1 or 22-1, the antigenic epitope region of the VP2 protein it identifies is 341-361 aa. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is an electrophoresis diagram of bacterial culture PCR amplification of the SCBV VP2 gene sequence in Example 1 of this invention. M represents the DNA standard DL8000, lanes 1-10 represent SCBV VP2 gene amplification, and lane 11 is the negative control.

[0020] Figure 2 This is an SDS-PAGE identification result of SCBV VP2 recombinant protein expression in Example 1 of this invention. M represents the protein molecular weight standard. Lane 1 is the pET28a empty vector; Lane 2 is the supernatant of SCBV VP2 recombinant protein after IPTG induction via ultrasonic disruption; Lane 3 is the precipitate of SCBV VP2 recombinant protein after IPTG induction via ultrasonic disruption; Lane 4 is the supernatant of uninduced SCBV VP2 recombinant protein after ultrasonic disruption; and Lane 5 is the precipitate of uninduced SCBV VP2 recombinant protein after ultrasonic disruption via ultrasonic disruption.

[0021] Figure 3 This is the purification and identification result of the SCBV VP2 recombinant protein in Example 1 of this invention. Where M is the molecular weight standard of the protein, lane 1 is the flow-through buffer, lane 2 is the washing buffer, and lanes 4-11 are the elution buffer for the purified protein.

[0022] Figure 4 This is the serum titer identification result of mice immunized with SCBV VP2 recombinant protein in Example 2 of the present invention.

[0023] Figure 5 This is the Western blotting analysis result of the monoclonal antibody in Example 2 of this invention. M represents the protein molecular weight standard, and lane 1 contains the pET28a empty vector protein, pET28a-G recombinant protein, and pET28a-VP2 recombinant protein.

[0024] Figure 6 This is the titer determination result of the monoclonal antibody in Example 2 of the present invention.

[0025] Figure 7 A is a schematic diagram of amino acid sequence truncation based on the VP2 protein; Figure 7 B represents the Western blotting analysis results of the overlapping truncated fragments, where M represents the protein molecular weight standard. Lane 1 is VP2 protein, lane 2 is truncated protein P1, lane 3 is truncated protein P2, lane 4 is truncated protein P3, lane 5 is truncated protein P4, lane 6 is truncated protein P5, lane 7 is truncated protein P6, lane 8 is truncated protein P7, lane 9 is truncated protein P8, lane 10 is truncated protein P9, lane 11 is truncated protein P10, and lane 12 is pGEX4T-1 empty vector protein.

[0026] The present invention will be further described below with reference to the accompanying drawings and description. Detailed Implementation

[0027] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain embodiments of the present invention, and should not be construed as limiting the present invention.

[0028] This invention provides a monoclonal antibody against the VP2 protein of a two-segmented RNA virus in mandarin fish, its preparation method, and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The method and application of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate changes and combinations to the method and application described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0029] The method for preparing a monoclonal antibody against the VP2 protein of a two-segmented RNA virus in mandarin fish provided by this invention includes: S1. The VP2 gene of SCBV was amplified and cloned into pET-28a(+) to obtain the recombinant plasmid pET28a-VP2. Then, the recombinant plasmid pET28a-VP2 was transformed into competent cells for expression and purified to obtain the VP2 protein. The specific steps for transforming the recombinant plasmid pET28a-VP2 into competent cells for expression are as follows: The recombinant plasmid pET28a-VP2 was transformed into BL21(DE3) competent cells, and single colonies were picked and cultured. The bacterial culture was then inoculated into LB medium containing kanamycin resistance and cultured with shaking until OD. 600 When the concentration was 0.6, IPTG solution was added to induce protein expression; The conditions for culturing single colonies were as follows: overnight culture at 37℃ and 220 rpm; the conditions for inoculating the bacterial culture into LB medium containing kanamycin resistance and shaking culture were as follows: the bacterial culture was inoculated into LB medium containing kanamycin resistance at a ratio of 1:100 and cultured at 37℃ and 220 rpm for 4 hours with shaking; the conditions for inducing protein expression by adding IPTG solution were as follows: protein expression was induced by adding IPTG solution to a final concentration of 1 mM and cultured at 37℃ and 220 rpm for 6 hours.

[0030] The purification steps are as follows: Collect the bacterial cells after expression culture, resuspend the cells in PBS, sonicate and centrifuge to collect the precipitate, lyse with lysis buffer and collect the supernatant solution for column chromatography purification.

[0031] S2. The purified VP2 protein was emulsified with Freund's adjuvant and immunized with 6-week-old female BALB / c mice. After immunization, blood was collected from the tail vein of the mice and serum was separated. The antibody titer of the immunized mice was detected by indirect ELISA. The mice with the highest titer were selected for intraperitoneal injection of VP2 protein for booster immunization. The specific steps for immunizing mice are as follows: The purified VP2 protein was emulsified with Freund's complete adjuvant at a 1:1 ratio at 100 μg / mouse and injected subcutaneously at multiple sites on the back of mice for immunization. Two weeks later, VP2 protein was emulsified with Freund's incomplete adjuvant at a 1:1 ratio for a second immunization. Two weeks later, a third immunization was performed. One week later, blood was collected from the tail vein of the immunized mice.

[0032] The specific steps for detecting serum antibody titers in immunized mice using the indirect ELISA method are as follows: 100 ng / well of purified VP2 protein was coated onto an ELISA plate overnight at 4°C. The plate was then blocked with 5% skim milk powder at 37°C for 2 h. The plate was washed with PBST buffer. Serum from immunized mice was diluted proportionally and used as the primary antibody. The plate was incubated at 37°C for 1 h. The plate was washed with PBST buffer. HRP-labeled goat anti-mouse antibody diluted 1:5000 was used as the secondary antibody and incubated at 37°C for 30 min. The plate was washed with PBST buffer. 100 μL / well of chromogenic solution was added and the plate was incubated at 37°C for 10 min. Finally, 50 μL / well of stop solution was added, and the data for each well were read using a microplate reader.

[0033] S3. Spleen cells from mice after booster immunization were collected and fused with SP2 / 0 myeloma cells. When the confluence of fused cells reached more than 50%, positive hybridoma cells secreting VP2 antibody were screened by indirect ELISA. The positive hybridoma cells were subcloned to obtain a hybridoma cell line that stably secretes monoclonal antibody against mandarin fish two-segment RNA virus VP2 protein. The specific steps involved in fusing spleen cells from booster-immunized mice with SP2 / 0 myeloma cells are as follows: After booster immunization, spleen cells from mice were fused with SP2 / 0 cells at a ratio of 5:1. 50% PEG1450 was added after 1 min, and the mixture was stirred for 2 min. 10 mL of preheated culture medium was added within 10 min. The mixture was incubated at 37°C for 5 min, centrifuged at 200 x g for 7 min, and then mixed into HAT medium containing 20% ​​FBS. 100 μL / well was added to 96-well dishes pre-coated with feeder cells until the confluence of cells to be successfully fused reached more than 50%.

[0034] Specifically, the subcloning of positive hybridoma cells involved four subcloning processes using the limiting dilution method to obtain four hybridoma cell lines with antibodies against the VP2 protein of the mandarin fish two-segment RNA virus.

[0035] S4. Eight-week-old female BALB / c mice were selected and injected intraperitoneally with Freund's incomplete adjuvant. Seven days later, the hybridoma cell line described above was injected, at a dose of 1×10⁻⁶. 6 Each mouse was tested until its abdomen swelled, then ascites was extracted, centrifuged, and purified to obtain a monoclonal antibody against the VP2 protein of the mandarin fish two-segment RNA virus.

[0036] For the monoclonal antibody against the bisegmental RNA virus VP2 protein of mandarin fish in this invention, the monoclonal antibody against the bisegmental RNA virus VP2 protein of mandarin fish is prepared by the preparation method of the monoclonal antibody against the bisegmental RNA virus VP2 protein of mandarin fish as described above.

[0037] Regarding the application of the monoclonal antibody against the bisegmental RNA virus VP2 protein of mandarin fish in this invention, as described above, it is used in identifying the antigenic epitopes of the VP2 protein and in preparing SCBV detection and diagnostic products.

[0038] For the above applications, when the monoclonal antibody against the bisegmental RNA virus VP2 protein of mandarin fish is monoclonal antibody 2-1, the antigenic epitope region of the VP2 protein it identifies is 1-25 aa; when the monoclonal antibody against the bisegmental RNA virus VP2 protein of mandarin fish is monoclonal antibody 13-1, the antigenic epitope region of the VP2 protein it identifies is 280-300 aa; when the monoclonal antibody against the bisegmental RNA virus VP2 protein of mandarin fish is monoclonal antibody 20-1 or 22-1, the antigenic epitope region of the VP2 protein it identifies is 341-361 aa.

[0039] The reagents and consumables used in this invention are all commercially available products. The invention is further illustrated below with reference to the embodiments: Example 1: Preparation of SCBV VP2 protein 1. Design of specific primers Based on the VP2 gene sequence of segment A of SCBV-FS202308 strain in NCBI, a pair of specific primers was designed for VP2 gene amplification. The primers were synthesized by Suzhou Genewise Biotechnology Co., Ltd. The designed primers are as follows: VP2-F: 5′-CATGGGCAGCAGCGAATTCACCGTAGTACCATACCTAAAATCCCTCA-3′ (SEQ ID NO. 2); VP2-R: 5'-GTGGTGGTGCTCGAGTCAATGGTGATGGTGATGGTGATGGTGTCTCAGAAATCCAAGG-3' (SEQ ID NO. 3).

[0040] 2. Construction of SCBV VP2 recombinant plasmid The VP2 gene was amplified by PCR using the primers (VP2-F, VP2-R) described above, with the pUC-GW-VP2 plasmid synthesized by Suzhou Genewiz Biotechnology Co., Ltd. as a template. The amplification system is shown in Table 1. Table 1. Content of each component in the PCR reaction system

[0041] The VP2 gene was amplified to a length of 1266 bp. The reaction program was as follows: pre-denaturation at 98℃ for 1 min; cycling: 98℃ for 10 s, 65℃ for 5 s, 72℃ for 10 s, 35 cycles; extension at 72℃ for 1 min. The nucleotide sequence of the VP2 gene is shown in SEQ ID NO.1.

[0042] The VP2 gene was cloned into the pET-28a(+) vector using homologous recombinase and named pET28a-VP2. The pET28a-VP2 recombinant plasmid was transformed into BL21(DE3) competent cells to obtain pET28a-VP2 recombinant bacterial culture. The pET28a-VP2 recombinant bacterial culture was identified by PCR. The PCR reaction system of the bacterial culture is shown in Table 2. The reaction program was as follows: 96℃ pre-denaturation for 2 min; cycling: 96℃ for 30 s, 65℃ for 30 s, 72℃ for 1 min, 35 cycles; extension at 72℃ for 5 min.

[0043] Table 2 Content of each component in bacterial PCR reaction

[0044] The results are as follows Figure 1 As shown, the target band of about 1266 bp was amplified, which is consistent with the expectation. Sequencing confirmed that the pET28a-VP2 recombinant plasmid was successfully constructed.

[0045] 3. Inducible expression of SCBV VP2 recombinant protein The pET28a-VP2 recombinant plasmid was transformed into BL21(DE3) competent cells. Single colonies were picked and cultured overnight at 37°C and 220 rpm as primary seed culture. The primary seed culture was diluted 1:100 in LB medium containing kanamycin resistance and cultured at 37°C and 220 rpm for 4 hours. IPTG solution was then added to a final concentration of 1 mM to induce protein expression, and the culture was continued at 37°C and 220 rpm for another 6 hours. The cells were collected by centrifugation, resuspended in PBS, and sonicated. After centrifugation at 12000 rpm for 10 min, the supernatant and precipitate were collected separately for SDS-PAGE analysis. Results are as follows: Figure 2 As shown, the SCBV VP2 recombinant protein is mainly expressed in the form of inclusion bodies, and its protein size is consistent with the theoretical molecular weight.

[0046] 4. Protein purification Following the Beyotime inclusion body protein purification kit protocol, add His-tag gel to an empty affinity chromatography column tube, discard the preservation solution, add denaturing lysis buffer to equilibrate the gel, discard the solution, and repeat twice. Seal the bottom of the column, add the inclusion body solution that has been pre-lysed with denaturing lysis buffer, seal the column, and incubate overnight at 4°C with shaking. The next day, open the bottom cap of the column, and allow the liquid to flow out from the bottom under gravity. Collect 80 ml of the solution. L of flow-through buffer was used for subsequent analysis; 20 mL of denaturing wash buffer was added to wash away contaminating proteins, and 80 μL of the solution was collected. The wash buffer effluent was used for subsequent analysis; elution was performed 5-10 times with denaturing elution buffer, 1.5 mL each time. The eluted protein was aliquoted into different centrifuge tubes, collecting 80 μL from each tube. L elution buffer was used for subsequent analysis. The collected samples were added to 5× protein loading buffer and mixed well. The mixture was then heated in a metal bath for 15 min, followed by SDS-PAGE protein electrophoresis to verify the purification effect. The results are as follows: Figure 3 As shown, the purified protein showed a single band at 47 kDa, indicating good purification results.

[0047] 5. Protein dialysis refolding The VP2 protein was refolded using dialysis bags, and the specific procedure is as follows: (1) Cut the dialysis bag to the required length according to the amount of protein (approximately 14cm is needed for 10mL of protein).

[0048] (2) Clean a 2L container, add an appropriate amount of ddH2O, put the cut dialysis bag into the container, and boil for 15 minutes.

[0049] (3) Cool down the dialysis bag and wash it 2-3 times with ddH2O.

[0050] (4) One person clamps one end and opens the other end, while another person carefully adds the protein into the open end with a pipette, removes air bubbles and clamps the open end. The clamped dialysis bag is placed in a container with 6 mol / L urea TGE and dialyzed at 4°C for 5-8 hours.

[0051] (5) Discard the 6 mol / L urea TGE in the beaker and add 4 mol / L urea TGE. Dialyze at 4°C for 12 h.

[0052] (6) Discard the 4 mol / L urea TGE in the beaker and replace it with 2 mol / L urea TGE. Dialyze at 4°C for 12 hours.

[0053] (7) Replace the 2 mol / L urea TGE in the beaker with 0 mol / L urea TGE and dialyze at 4°C for 12 hours.

[0054] (8) Discard the 0 mol / L urea TGE in the beaker and replace it with PBS. Dialyze at 4°C for 12 hours.

[0055] (9) The protein after dialysis was preserved and its concentration was determined to be 1.2 mg / mL.

[0056] For the dialysis solution (TGE) with 6 mol / L urea: add various reagents according to Table 3, then add 360.36 g of urea, add ddH2O to 1000 mL, mix evenly with a magnetic stirrer, adjust the pH to 8.0 with HCl, and place at 4℃.

[0057] Dialysis solution (TGE) with 4 mol / L urea: Add all reagents according to Table 3, then add 240.24 g of urea, add ddH2O to 1000 mL, mix well with a magnetic stirrer, adjust the pH to 8.0 with HCl, and place at 4℃.

[0058] Dialysis solution (TGE) with 2 mol / L urea: Add all reagents according to Table 3, then add 120.12 g of urea, add ddH2O to 1000 mL, mix well with a magnetic stirrer, adjust the pH to 8.0 with HCl, and place at 4℃.

[0059] Dialysis solution (TGE) with 0 mol / L urea: Add all reagents according to Table 3, then add 0 g urea, add ddH2O to 1000 mL, mix well with a magnetic stirrer, adjust pH to 8.0 with HCl, and place at 4℃.

[0060] Table 3 Dialysis fluid formulation

[0061] Example 2: Preparation of SCBV VP2 monoclonal antibody 1. Mouse immunization The purified VP2 protein was emulsified with Freund's complete adjuvant at a 1:1 ratio of 100 μg / mouse and injected subcutaneously at multiple sites on the back of the mice for immunization. Two weeks later, the mice were emulsified with VP2 protein and Freund's incomplete adjuvant at a 1:1 ratio for a second immunization. Two weeks later, the mice were immunized for a third immunization. One week later, blood was collected from the tail vein of the immunized mice.

[0062] 2. Detection of serum titer in immunized mice using indirect ELISA method 100 ng / well of purified VP2 protein was coated onto ELISA plates overnight at 4°C. The plates were then blocked with 5% skim milk at 37°C for 2 h. The plates were washed with PBST buffer. Serum from immunized mice was diluted proportionally and used as the primary antibody, incubated at 37°C for 1 h. The plates were washed with PBST buffer again. HRP-labeled goat anti-mouse antibody (1:5000 dilution) was used as the secondary antibody and incubated at 37°C for 30 min. The plates were washed with PBST buffer again, and 100 μL / well of chromogenic buffer was added and incubated at 37°C for 10 min. Finally, 50 μL / well of stop solution was added, and the data from each well were read using a microplate reader. Results are as follows: Figure 4 As shown, the mice had high serum titers, so mice with the highest serum titers were selected and injected intraperitoneally with 100 μg of protein to enhance immunization.

[0063] 3. Cell fusion Spleen cells from mice after booster immunization were fused with SP2 / 0 cells at a ratio of 5:1. 50% PEG1450 was added over 1 minute, and the mixture was stirred for 2 minutes. 10 mL of pre-warmed culture medium was added over 10 minutes, and the mixture was incubated at 37°C for 5 minutes. After centrifugation at 200xg for 7 minutes, the mixture was transferred to HAT medium containing 20% ​​FBS. 100 μL / well was added to 96-well dishes pre-coated with feeder cells. Once the confluence of successfully fused cells reached over 50%, positive hybridoma cell lines secreting VP2 antibodies were screened using an indirect ELISA method. The screened positive hybridoma cells were subcloned four times using a limiting dilution method to obtain four monoclonal cell lines secreting VP2 antibodies.

[0064] 4. Identification of monoclonal antibody subclasses Supernatant from positive monoclonal cells after four rounds of subclonal screening was collected, and the monoclonal antibody subclass was determined using the Biodrogon mouse monoclonal antibody Ig class / subclass identification ELISA kit. Specific methods can be found in the kit's instruction manual. The results are shown in Table 4. The heavy chain type of antibody secreted by cell line 2-1 was IgG2b, while the heavy chain type of antibody secreted by cell lines 13-1, 20-1, and 22-1 was IgG1. The light chain type of antibody secreted by all four cell lines was Kappa.

[0065] Table 4. Identification results of monoclonal antibody subclasses

[0066] If the OD450 value of the test sample is greater than the OD450 value of the negative sample + 0.15, it is judged as positive; if the OD450 value of the negative sample is less than 0.05, it is calculated as 0.05.

[0067] 5. Preparation of ascites Ascites fluid was prepared from positive hybridoma cells with strong antibody secretion and good growth status via in vivo induction. Female BALB / c mice were sensitized with Freund's incomplete adjuvant, and 500 μL was injected intraperitoneally. The cells were then ready for use after 7 days. The cells were removed from the dish, washed twice with culture medium, and the cell count was adjusted to 1 × 10⁻⁶. 6 Cells were injected intraperitoneally into sensitized mice at a concentration of 1 cell / mL. After 1–2 weeks, when the mice’s abdomens were significantly distended, the ascites was drained, centrifuged at 12,000 r / min for 15 min, and the supernatant was collected, aliquoted, and stored at -80°C.

[0068] 6. Western blotting analysis of monoclonal antibodies Western blotting was performed on the prepared ascites fluid. pET28a-VP2 recombinant protein, pET28a-G, and pET-28a(+) empty vector protein were used as antigens. The obtained ascites fluid was diluted 1:5000 as the primary antibody, and goat anti-mouse IgG was diluted 1:5000 as the secondary antibody. Results are as follows: Figure 5 As shown, the pET28a-VP2 recombinant protein can specifically bind to the prepared ascites fluid and show obvious bands, but it does not react with the pET28a-G and pET-28a(+) empty vector proteins, indicating that the four monoclonal antibodies prepared have good specificity.

[0069] 7. Monoclonal antibody titer determination The prepared ascites fluid was diluted at ratios of 1:100, 1:500, 1:1000, 1:5000, 1:10000, 1:5000, 1:100000, 1:500000, and 1:1000000. A negative mouse serum control was also included. Antibody titers were detected using an indirect ELISA method. Results are as follows: Figure 6 As shown, the ascites titers of 13-1 and 22-1 reached 1:500000, and the ascites titers of 2-1 and 20-1 reached 1:1000000.

[0070] 8. Identification of recognition epitopes for monoclonal antibodies First, VP2 (422aa) was divided into three segments (P1: 1-150aa; P2: 130-280aa; P3: 260-422aa). Expression plasmids were constructed using the primers (SEQ NO: 6-11) in Table 5. A schematic diagram of the segmentation is shown below. Figure 7 A. The target fragment was ligated into the pGEX4T-1 vector for induced expression. Samples were prepared after ultrasonic disruption and analyzed by Western blotting. Results are as follows: Figure 7 As shown in B, the 2-1 monoclonal antibody recognizes P1 (1-150 aa), while the 13-1, 20-1, and 22-1 monoclonal antibodies recognize P3 (260-422 aa). Therefore, P1 and P3 were further truncated into three segments (P4: 75-341 aa; P5: 95-320 aa; P6: 115-300 aa). Expression plasmids were constructed using the primers (SEQ NO: 12-17) in Table 5, and expression induction was performed as described above. The results are as follows: Figure 7 As shown in B, the recognition epitope of the 13-1 monoclonal antibody is 280-300 aa, the recognition region of the 2-1 monoclonal antibody is 1-75 aa, and the recognition epitopes of the 20-1 and 22-1 monoclonal antibodies are 341-422 aa. Therefore, P1 and P3 were further truncated into four segments (P7: 55-361 aa; P8: 35-381 aa; P9: 25-401 aa; P10: 15-411 aa). Expression plasmids were constructed using the primers (SEQ NO: 18-25) in Table 5, and expression induction was performed as described above. The results are as follows: Figure 7 As shown in B, the recognition epitope of the 2-1 monoclonal antibody is 1-25aa, and the recognition epitopes of the 20-1 and 22-1 monoclonal antibodies are 341-361aa.

[0071] Table 5 Primer sequences in this invention

[0072] 9. PCR amplification and sequencing of the variable region gene of cloning antibody After culturing the selected cell lines 2-1, 13-1, 20-1, and 22-1, total RNA was extracted and reverse transcribed to amplify the heavy and light chain genes. These genes were then ligated into the pMD19-T vector, transformed into competent cells, and plasmids were extracted and sent to Guangzhou Qingke Biotechnology Co., Ltd. for sequencing. The results showed that the amino acid sequences of the complementarity-determining regions (CDR1-CDR3) of the 2-1 heavy chain variable region were: GYTFTEVA, FNPNNGDT, TRTFDY (SEQ ID NO.26-SEQ ID NO.28); the amino acid sequences of the complementarity-determining regions (CDR1-CDR3) of the 2-1 light chain variable region were: QSVSND, YTS, QQDHSSPWT (SEQ ID NO.29-SEQ ID NO.31); and the amino acid sequences of the complementarity-determining regions (CDR1-CDR3) of the 13-1 heavy chain variable region were: GYTFTSYW, IDPSDNET, SMVSVYFTNL (SEQ ID NO.32-SEQ ID NO.28). The amino acid sequences of the complementarity-determining regions (CDR1-CDR3) of the 13-1 light chain variable region are: QSIGTS, YAS, QQSNSWPLT (SEQ ID NO. 35-SEQ ID NO. 37); the amino acid sequences of the complementarity-determining regions (CDR1-CDR3) of the 20-1 heavy chain variable region are: GFNIQDDY, IDPANGIT, AIYYYGSSYYFDY (SEQ ID NO. 38-SEQ ID NO. 40); the amino acid sequences of the complementarity-determining regions (CDR1-CDR3) of the 20-1 light chain variable region are: MTMITPSLHAC, KVS, FQGSHVPLT (SEQ ID NO. 41-SEQ ID NO. 43); and the amino acid sequences of the complementarity-determining regions (CDR1-CDR3) of the 22-1 heavy chain variable region are: GYIFTSYW, IDPTDSET, ARGMRFAY (SEQ ID NO. 44-SEQ ID NO. 37). The amino acid sequences of the complementarity-determining regions (CDR1-CDR3) of the 22-1 light chain variable region (SEQ ID NO.46) are: KSVSTSGYSY, PVS, QHIRELTR (SEQ ID NO.47-SEQ ID NO.49).

[0073] In summary, this invention provides a monoclonal antibody against the SCBV VP2 protein of mandarin fish, its preparation method, and its application. The invention involves cloning the SCBV VP2 gene sequence into a pET-28a(+) vector to construct a His-tag-enabled prokaryotic expression vector capable of expressing the SCBV VP2 protein. This vector is then transformed into competent cells, and the SCBV VP2 protein is obtained after induction of expression and purification using the His-tag tag. The purified protein was used as an immunogen to immunize Balb / c mice. Monoclonal antibodies against SCBV VP2 were obtained through cell fusion and subclonal selection, and their variable region sequence was identified. Subtype identification results showed that the heavy chain type of the antibody secreted by cell line 2-1 was IgG2b, the heavy chain type of the antibody secreted by cell lines 13-1, 20-1, and 22-1 was IgG1, and the light chain type of the antibody secreted by all four cell lines was Kappa. Western blotting analysis showed that the obtained monoclonal antibody specifically recognizes the SCBV VP2 protein. After identification, the recognition epitope of the 2-1 monoclonal antibody was 1-25aa, the recognition epitope of the 13-1 monoclonal antibody was 280-300aa, and the recognition epitope of the 20-1 and 22-1 monoclonal antibodies was 341-361aa, further demonstrating the specificity of the antibody.

[0074] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention are equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. A method for preparing a monoclonal antibody against the VP2 protein of a two-segmented RNA virus in mandarin fish, characterized in that, The method includes: S1. The VP2 gene of SCBV was amplified and cloned into pET-28a(+) to obtain the recombinant plasmid pET28a-VP2. Then, the recombinant plasmid pET28a-VP2 was transformed into competent cells for expression and purified to obtain the VP2 protein. S2. The purified VP2 protein was emulsified with Freund's adjuvant and immunized with 6-week-old female BALB / c mice. After immunization, blood was collected from the tail vein of the mice and serum was separated. The antibody titer of the immunized mice was detected by indirect ELISA. The mice with the highest titer were selected for intraperitoneal injection of VP2 protein for booster immunization. S3. Spleen cells from mice after booster immunization were collected and fused with SP2 / 0 myeloma cells. When the confluence of fused cells reached more than 50%, positive hybridoma cells secreting VP2 antibody were screened by indirect ELISA. The positive hybridoma cells were subcloned to obtain a hybridoma cell line that stably secretes monoclonal antibody against mandarin fish two-segment RNA virus VP2 protein. S4. Eight-week-old female BALB / c mice were selected and injected intraperitoneally with Freund's incomplete adjuvant. Seven days later, the hybridoma cell line described above was injected, at a dose of 1×10⁻⁶. 6 Each mouse was tested until its abdomen swelled, then ascites was extracted, centrifuged, and purified to obtain a monoclonal antibody against the VP2 protein of the mandarin fish two-segment RNA virus.

2. The method for preparing a monoclonal antibody against the VP2 protein of a two-segmented RNA virus in mandarin fish according to claim 1, characterized in that, In step S1, the specific steps of transforming the recombinant plasmid pET28a-VP2 into competent cells for expression are as follows: The recombinant plasmid pET28a-VP2 was transformed into BL21(DE3) competent cells, and single colonies were picked and cultured. The bacterial culture was then inoculated into LB medium containing kanamycin resistance and cultured with shaking until OD. 600 When the concentration was 0.6, IPTG solution was added to induce protein expression; The conditions for culturing single colonies were as follows: overnight culture at 37℃ and 220 rpm; the conditions for inoculating the bacterial culture into LB medium containing kanamycin resistance and shaking culture were as follows: the bacterial culture was inoculated into LB medium containing kanamycin resistance at a ratio of 1:100 and cultured at 37℃ and 220 rpm for 4 hours with shaking; the conditions for inducing protein expression by adding IPTG solution were as follows: protein expression was induced by adding IPTG solution to a final concentration of 1 mM and cultured at 37℃ and 220 rpm for 6 hours.

3. The method for preparing a monoclonal antibody against the VP2 protein of a two-segmented RNA virus in mandarin fish according to claim 1, characterized in that, In step S1, the purification step specifically includes: Collect the bacterial cells after expression culture, resuspend the cells in PBS, sonicate and centrifuge to collect the precipitate, lyse with lysis buffer and collect the supernatant solution for column chromatography purification.

4. The method for preparing a monoclonal antibody against the VP2 protein of a two-segmented RNA virus in mandarin fish according to claim 1, characterized in that, In step S2, the specific steps for immunizing the mice are as follows: The purified VP2 protein was emulsified with Freund's complete adjuvant at a 1:1 ratio at 100 μg / mouse and injected subcutaneously at multiple sites on the back of mice for immunization. Two weeks later, VP2 protein was emulsified with Freund's incomplete adjuvant at a 1:1 ratio for a second immunization. Two weeks later, a third immunization was performed. One week later, blood was collected from the tail vein of the immunized mice.

5. The method for preparing a monoclonal antibody against the VP2 protein of a two-segmented RNA virus in mandarin fish according to claim 1, characterized in that, In step S2, the specific steps for detecting the antibody titer in the serum of immunized mice using the indirect ELISA method are as follows: 100 ng / well of purified VP2 protein was coated onto an ELISA plate overnight at 4°C. The plate was then blocked with 5% skim milk powder at 37°C for 2 h. The plate was washed with PBST buffer. Serum from immunized mice was diluted proportionally and used as the primary antibody. The plate was incubated at 37°C for 1 h. The plate was washed with PBST buffer. HRP-labeled goat anti-mouse antibody diluted 1:5000 was used as the secondary antibody and incubated at 37°C for 30 min. The plate was washed with PBST buffer. 100 μL / well of chromogenic solution was added and the plate was incubated at 37°C for 10 min. Finally, 50 μL / well of stop solution was added, and the data for each well were read using a microplate reader.

6. The method for preparing a monoclonal antibody against the VP2 protein of a two-segmented RNA virus in mandarin fish according to claim 1, characterized in that, In step S3, the specific steps for fusing spleen cells from mice after booster immunization with SP2 / 0 myeloma cells are as follows: After booster immunization, spleen cells from mice were fused with SP2 / 0 cells at a ratio of 5:

1. 50% PEG1450 was added after 1 min, and the mixture was stirred for 2 min. 10 mL of preheated culture medium was added within 10 min. The mixture was incubated at 37°C for 5 min, centrifuged at 200 x g for 7 min, and then mixed into HAT medium containing 20% ​​FBS. 100 μL / well was added to 96-well dishes pre-coated with feeder cells until the confluence of cells to be successfully fused reached more than 50%.

7. The method for preparing a monoclonal antibody against the VP2 protein of a two-segmented RNA virus in mandarin fish according to claim 1, characterized in that, In step S3, the subcloning of positive hybridoma cells specifically involves performing four subclonings using the limiting dilution method to obtain four hybridoma cell lines with antibodies against the VP2 protein of the mandarin fish two-segment RNA virus.

8. A monoclonal antibody against the VP2 protein of a two-segmented RNA virus in mandarin fish, characterized in that, The monoclonal antibody against the bisegmental RNA virus VP2 protein of mandarin fish was prepared using the method described in any one of claims 1-7.

9. The application of the monoclonal antibody against the bisegmental RNA virus VP2 protein of mandarin fish as described in claim 8 in identifying the antigenic epitopes of the VP2 protein and preparing SCBV detection and diagnostic products.

10. The application according to claim 9, characterized in that, When the monoclonal antibody against the bisegmental RNA virus VP2 protein of mandarin fish is monoclonal antibody 2-1, the antigenic epitope region of the VP2 protein it identifies is 1-25 aa; when the monoclonal antibody against the bisegmental RNA virus VP2 protein of mandarin fish is monoclonal antibody 13-1, the antigenic epitope region of the VP2 protein it identifies is 280-300 aa; when the monoclonal antibody against the bisegmental RNA virus VP2 protein of mandarin fish is monoclonal antibody 20-1 or 22-1, the antigenic epitope region of the VP2 protein it identifies is 341-361 aa.

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